Physical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden.
J Phys Chem B. 2010 Jun 3;114(21):7229-44. doi: 10.1021/jp9119809.
Poly(amido amine) (PAMAM) dendrimers have previously been shown, as cationic condensing agents of DNA, to have high potential for nonviral gene delivery. This study addresses two key issues for gene delivery: the interaction of the biomembrane with (i) the condensing agent (the cationic PAMAM dendrimer) and (ii) the corresponding dendrimer/DNA aggregate. Using in situ null ellipsometry and neutron reflection, parallel experiments were carried out involving dendrimers of generations 2 (G2), 4 (G4), and 6 (G6). The study demonstrates that free dendrimers of all three generations were able to traverse supported palmitoyloleoylphosphatidylcholine (POPC) bilayers deposited on silica surfaces. The model biomembranes were elevated from the solid surfaces upon dendrimer penetration, which offers a promising new way to generate more realistic model biomembranes where the contact with the supporting surface is reduced and where aqueous cavities are present beneath the bilayer. The largest dendrimer (G6) induced partial bilayer destruction directly upon penetration, whereas the smaller dendrimers (G2 and G4) leave the bilayer intact, so we propose that lower generation dendrimers have greater potential as transfection mediators. In addition to the experimental observations, coarse-grained simulations on the interaction between generation 3 (G3) dendrimers and POPC bilayers were performed in the absence and presence of a bilayer-supporting negatively charged surface that emulates the support. The simulations demonstrate that G3 is transported across free-standing POPC bilayers by direct penetration and not by endocytosis. The penetrability was, however, reduced in the presence of a surface, indicating that the membrane transport observed experimentally was not driven solely by the surface. The experimental reflection techniques were also applied to dendrimer/DNA aggregates of charge ratio = 0.5, and while G2/DNA and G4/DNA aggregates interact with POPC bilayers, G6/DNA displays no such interaction. These results indicate that, in contrast to free dendrimer molecules, dendrimer/DNA aggregates of low charge ratios are not able to traverse a membrane by direct penetration.
聚（酰胺-胺）（PAMAM）树状大分子以前被证明作为 DNA 的阳离子缩合剂，具有很高的非病毒基因传递潜力。本研究解决了基因传递的两个关键问题：（i）生物膜与缩合剂（阳离子 PAMAM 树状大分子）的相互作用和（ii）相应的树状大分子/DNA 聚集体与生物膜的相互作用。本研究使用原位无椭圆偏振和中子反射，进行了涉及第二代 (G2)、第四代 (G4) 和第六代 (G6) 树状大分子的平行实验。研究表明，所有三代的游离树状大分子都能够穿透沉积在硅表面上的棕榈酰油酰磷脂酰胆碱 (POPC) 双层。当树状大分子穿透时，模型生物膜从固体表面抬起，这为生成更真实的模型生物膜提供了一种有前途的新方法，在这种模型生物膜中，与支撑表面的接触减少，双层下面存在水腔。最大的树状大分子 (G6) 直接穿透时导致部分双层破坏，而较小的树状大分子 (G2 和 G4) 则使双层保持完整，因此我们提出较低代的树状大分子具有更大的作为转染介导物的潜力。除了实验观察结果外，还在不存在和存在模拟支撑的带负电荷表面的情况下，对第三代 (G3) 树状大分子与 POPC 双层之间的相互作用进行了粗粒化模拟。模拟表明，G3 通过直接穿透而不是内吞作用穿过独立的 POPC 双层。然而，在存在表面的情况下，穿透性降低，这表明实验中观察到的膜传输不是仅由表面驱动的。实验反射技术也应用于电荷比 = 0.5 的树状大分子/DNA 聚集体，虽然 G2/DNA 和 G4/DNA 聚集体与 POPC 双层相互作用，但 G6/DNA 则没有这种相互作用。这些结果表明，与游离树状大分子分子相反，低电荷比的树状大分子/DNA 聚集体不能通过直接穿透来穿透膜。
